The long-term goal of this project is to elucidate the detailed mechanism of action on a molecular level of the enzymes of the blood coagulation and fibrinolytic pathways, especially by using low molecular weight substrates and inhibitors. This research focuses on human thrombin, plasmin and factor Xa. We shall study the specificity (active and primary binding sites) of these enzymes using peptide and other synthetic substrates and irreversible (affinity-labeling agents) and reversible (including transition-state) inhibitors. Bovine beta-trypsin is used as a standard of comparison. Methods for the isolation and purification of human factor Xa from Cohn fraction III will also be investigated. These include classical methods, the use of hydrophobic chromatography, and methods exploiting the metal-binding properties of the factor Xa zymogen.